Carbohydrates are added and further modified to proteins passing through the secretory pathway. The enzymes responsible for these additions and modifications span the membranes and extend in the lumen of the protein processing centers of the cell, the endoplasmic reticulum and Golgi apparatus. In general, glycosylation is important for either maintaining or promoting protein stability, or enabling interactions with carbohydrate-specific receptors. Most proteins produced in our bodies are glycosylated, or modified by the attachment of sugars, include soluble proteins in circulation and receptors on the surface of cells. The importance of glycosylation in health and disease is increasingly appreciated. Many successful pathogens, including HIV and Mycobacterium tuberculosis, cover themselves in glycans to hide from the immune system. Stem cell trafficking is controlled by glycosylation. The red blood cell ABO types are determined by glycosylation. Also, immunoglobulin effector functions are controlled by glycosylation. Surprisingly, little is known about the regulation of glycosylation, despite the many distinct biological roles it plays. Further attempts to manipulate glycosylation have used crude, in vitro systems, which are limited in scope and applicability. The molecular biology revolution that drives medicine forward has overshadowed these deficiencies in glycobiology. Intriguing, many of the secretory pathway enzymes that attach and alter glycans are secreted into the circulation. This proposal aims exploit the natural soluble forms of glycosylation enzymes, enabling glycoengineering of soluble proteins and surface receptors in vivo. These studies will enable examination of specific glycan modification. Further, we will explore the therapeutic potential of glycoengineering. For example, glycans involved in HIV and Influenza pathogenesis will be altered to curb infections, antibodies will be engineered in the circulation, and all red blood cells will be converted into universal typ O donors. Thus, this proposal will decipher the biological contribution of specific glycans on individual proteins, potentially impacting a range of disease types.